PROJECT SUMMARY/ABSTRACT Distant metastases cause >90% of cancer-related mortality. In osteosarcoma, metastatic progression typically occurs as the cancer spreads from the bones to the lungs. Forty percent of patients with osteosarcoma die from metastatic disease. There is an urgent clinical need to determine the factors responsible for lung metastasis in osteosarcoma so that these factors can be exploited therapeutically. Our lab discovered that epigenetic alterations at enhancer elements play a critical role in the transition of normal cells to malignant cells during tumorigenesis. More recently, the lab has gathered evidence that enhancer alterations also contribute to metastasis, specifically in osteosarcoma. Through epigenomic studies of non-metastatic and highly lung-metastatic osteosarcoma cell line pairs, combined with functional studies, our lab homed in on a gene, Tissue Factor III (F3), that appears to be a critical regulator of the metastatic process in osteosarcoma. The F3 locus is endowed with multiple active enhancer elements in metastatic cells, and these enhancers are almost completely absent in non-metastatic cells. Through functional studies in vivo, we show that F3 is switched on as the metastatic cells engage the lung microenvironment. Remarkably, blocking the activation of F3 via shRNA dramatically impairs the ability of the cells to colonize and proliferate in the lungs, suggesting F3 activation is required for metastasis. Our aims in this study are twofold. First we will test the hypothesis that the enhancers acquired in the metastatic cells at the F3 locus are responsible for activating F3 expression in the lung. This hypothesis will be tested via direct genetic and epigenetic manipulation of the F3 enhancers using TALENs, CRISPR-Cas9, and CRISPRi. Second, we will use chromatin conformation capture technology (4C-seq) to test if the mechanistic basis of F3 gene activation is physical looping between the acquired enhancers and the F3 promoter. As part of this aim we will also test whether the potential contacts between the F3 enhancers and promoters in metastatic cells are absent from non-metastatic cells. Successful completion of these experiments would provide new evidence that non-coding DNA elements are critical a critical driver of metastatic progression of cancer.